Thousands of genomes have been sequenced, but the functions of most of the genes that they encode remain largely unknown. My lab aims to transform our ability to engineer biology by developing broadly applicable tools that dramatically accelerate the study of uncharacterized genes. In this New Innovator project, we will use chemical genetics to assign functions to thousands of uncharacterized genes in the green alga Chlamydomonas reinhardtii, a powerful model system central to studies of ciliary biogenesis and motility, centrosomes, photosynthesis, electron transport, inter-organelle communication and optogenetics. We will leverage game-changing tools developed by my lab, including the first genome-wide collection of indexed mutants in this organism and methods for tracking mutant abundances in pools of 100,000 mutants. We will quantify the growth defects and enhancements of mutants representing nearly all genes in the genome across 200 growth conditions. Additionally, in a thrust focusing on ciliary function, we will quantify ciliary phenotypes for all mutants in the presence of 50 recently discovered small molecule modulators of cilia function. We will use these data to accurately predict functions for thousands of uncharacterized genes based on the principle that mutants in genes that function in the same pathway usually show similar phenotypes. Furthermore, we will identify the genetic targets of the small molecule modulators of cilia function using chemical-genetic interactions. We will dissect gene functions in collaboration with the scientific community, with a focus on novel genes essential for cilia biogenesis. More broadly, the project will serve as a platform for advancing the development of cutting-edge tools to systematically and quantitatively probe gene functions throughout the tree of life.